Inhibition of epileptiform activity by neuropeptide Y in brain tissue from drug-resistant temporal lobe epilepsy patients

Abstract

In epilepsy patients, drug-resistant seizures often originate in one of the temporal lobes. In selected cases, when certain requirements are met, this area is surgically resected for therapeutic reasons. We kept the resected tissue slices alive in vitro for 48 h to create a platform for testing a novel treatment strategy based on neuropeptide Y (NPY) against drug-resistant epilepsy. We demonstrate that NPY exerts a significant inhibitory effect on epileptiform activity, recorded with whole-cell patch-clamp, in human hippocampal dentate gyrus. Application of NPY reduced overall number of paroxysmal depolarising shifts and action potentials. This effect was mediated by Y2 receptors, since application of selective Y2-receptor antagonist blocked the effect of NPY. This proof-of-concept finding is an important translational milestone for validating NPY-based gene therapy for targeting focal drug-resistant epilepsies, and increasing the prospects for positive outcome in potential clinical trials.

Conflict of interest statement

M.K. and D.W. are co-founders and consultants of spin-off company COMBIGENE AB, Sweden. M.K. and D.W. are also inventors on awarded patent pertaining to the data presented (WO2008004972).

Figures

Figure 1

Translational roadmap for the use of human brain tissue as a step between animal research and clinical trials. Schematic illustration of how human-tissue resected from patients with epilepsy can be used to validate the effect of treatments found in animal models before proceeding to clinical trials for drug-resistant epilepsy, exemplified by NPY. (1) Basic research, with in vitro models, from several labs has shown that NPY has an inhibitory effect on epileptiform activity. (2) Chronic epilepsy models in vivo, such as the intrahippocampal kainate model, has provided key evidence demonstrating that NPY has an anti-epileptic effect in the chronic epileptic brain over a longer period of time. (3) The present study is a crucial validation step showing that NPY has an inhibitory effect on epileptiform activity in the target tissue, drug-resistant human hippocampal slices, minimising the risk for the next step of first-in-man clinical phase 1–2 study (step 4). The step 2 is interchangeable with step 3.

Figure 2

Neuropathological evaluation and granule cell layer with biocytin-filled cell: Example of (a–c) H&E and (d–f) MAP2 staining in hippocampal tissue resected from a patient with TLE. The sectioning and staining are made for neuropathological evaluation showing (a,d) the whole slice including dentate gyrus, CA3-CA1 and subiculum. The electrophysiological experiments were performed in the dentate gyrus magnified in B and E with the granule cell layer (gcl) identified, and further magnification inset showing individual granule cells. (c,f) In CA1, the neuronal cell loss is clear, the pyramidal cell layer has completely disappeared. After electrophysiological experiments the slices were fixated in PFA and later stained for (g) MAP2 (red) showing the granule cell layer with the recorded biocytin-filled granule cell (in green). (h) Higher magnification of the recorded neuron with typical granule cell morphology. Scale bar: 2 mm in a and d, 150 µm in b, c, e and f with 50 µm in inset, 200 µm in g and 50 µm in h.

Figure 3

NPY-application suppresses epileptiform activity in human drug-resistant hippocampal slices. (a) Example recordings from dentate granule cells during epileptiform activity triggered by 0Mg2+/4-AP aCSF. Whole-cell recordings in current-clamp mode, arrows indicate the PDSs that are displayed in expanded time-scale to the right of each sweep. NPY application resulted in a marked reduction of APs and PDS frequencies compared to the baseline, and partial return to the baseline levels during the washout period. (b–d) The average values (±SEM) are displayed in orange, the individual values – in grey. (b) The number of PDS was reduced during the application of NPY (baseline: 47.42 ± 10.52 and NPY: 17.6 ± 6.06, n = 12, t-test, p = 0.0022). (c) The number of APs was reduced during the application of NPY (baseline: 1042 ± 205.1 and NPY: 371.8 ± 71.49, n = 12, t-test, p = 0.0013). (d) The number of APs during individual PDSs did not change during the application of NPY (baseline: 6.067 ± 1.66 and NPY: 7.94 ± 2.786, n = 12, t-test, p = 0.9436). (e) Cumulative plot of the time-interval between APs in baseline recordings (black) and recordings during NPY application (blue). A significant difference between baseline and NPY (p < 0.0001, D = 0.1833) was detected with the Kolmogorov-Smirnov test.

Figure 4

NPY application together with its Y2-receptor antagonist BIIE0246 has no effect on epileptiform activity. (a) Example recordings from dentate granule cells during epileptiform activity triggered by 0Mg2+/4-AP aCSF. Whole-cell recordings in current-clamp mode, arrows indicate the PDSs that are displayed in zoomed-in time-scale to the right of each sweep. NPY applied together with the Y2 receptor antagonist BIIE0246; under these conditions a reduction in AP or PDS frequency is not observed. (b–d) The average values (±SEM) are displayed in orange, the individual values – in grey. (b) The number of PDS is unchanged during the application of NPY + BIIE0246 (baseline: 35.75 ± 12.33 and NPY + BIIE0246: 26 ± 12.27, n = 8, t-test, p = 0.0834). (c) The number of APs is unchanged during the application of NPY + BIIE0246 (baseline: 633.6 ± 232.8 and NPY + BIIE0246: 650.4 ± 265.3, n = 9, t-test, p = 0.874). (d) The number of APs during the individual PDSs is unchanged during the application of NPY + BIIE0246 (baseline: 4.674 ± 1.028 and NPY + BIIE0246: 5.873 ± 1.361, n = 7, t-test, p = 0.094). (e) Cumulative plot of the time-interval between APs during the baseline recordings (black) and recordings during NPY + BIIE0246 application (blue). No difference between baseline and NPY + BIIE0246 (p = 0.2743, D = 0.0794) was detected with the Kolmogorov-Smirnov test.

Figure 5

Simultaneous whole-cell current-clamp and field-recordings in the dentate gyrus during NPY application. (a) Representative recordings showing simultaneous whole-cell current-clamp recording and a field recording during baseline recording, NPY application, and washout period. Whole-cell recording from individual granule cell demonstrating 4-AP induced PDSs that are time-locked with field potential bursting. Both PDS and corresponding field potential bursting showed parallel reductions in frequency during NPY application. (b) Representative recordings showing an unfiltered PDS (top), a PDS low-pass filtered at 40 Hz with the best fit indicated by the red line (middle) and a low-pass filtered at 40 Hz field potential (bottom). (c) Cross-correlation analysis of whole-cell and field recordings showing a significant degree of time correlation, with the cross-correlation function estimate peaking close to 0 ms. The red trace is the average correlation estimate across recordings, the grey shadow represents SEM. (d) The number of LFPs plotted against the number of PDSs for each simultaneous whole-cell and field recording with the calculated Deming regression line plotted. The correlation between the number of LFPs and the PDSs is significant (p = 0.0028, n = 7, r = 0.9643) and the calculated regression line intersects the x-axis at 10.5.